Stroke is the second leading cause of death and the leading cause of adult disability worldwide. Despite an impressive amount of neuroprotective agents that has been identified in experimental stroke, none of them proved efficient in clinical trials. There is a general consensus that an effective treatment requires the ability to interact with not one, but multiple pathophysiological cascades at different levels that induced by the insult – cocktail therapy. Luckily, recent progress in the field of epigenetics revealed that epigenetic modifications had influence on many known pathways involved in the complex course of ischemic disease development. The fact that epigenetic molecules, by altering transcriptional regulation, may simultaneously act on different levels of ischemic brain injury makes them promising candidates for clinical use. These modifications arise typically owing to deoxyribonucleic acid methylation and histone acetylation. The aim of this review is to give a comprehensive overview of current advances in stroke epigenetics, in particular, the physiological and pathological functions of the 11 classical histone deacetylases.

Stroke (cerebrovascular accident) is used as a genetic term for a clinical syndrome in the brain. In the United States, according to the National Institute of Health (NIH), stroke is the third leading cause of death and the leading cause of adult disability. Approximately 795,000 stroke incidences occur every year (including 610,000 new cases and 185,000 recurrences), meanwhile about 20% of stroke patients die. On average, every 40 s, someone in the United States has a stroke, and on average, every 4 min, someone dies of a stroke. It is estimated that around 6.4 million people in the USA suffered stroke today; 2.5 million are males and 3.9 million are females. Projections show that by 2030, an additional 3.4 million US adults aged ≥18 years will have had a stroke. Globally, there were 6.5 million stroke deaths in 2013, making stroke the second-leading cause of death behind ischemic heart disease.[1],[2] Generally, there are three treatment stages for stroke: prevention, therapy immediately after stroke, and poststroke rehabilitation. Therapies for stroke include medications, surgery, or rehabilitation. Up to now, besides tissue-type plasminogen activator (tPA), no other treatment to limit brain damage is yet available. Unfortunately, tPA has a short therapeutic window of 3–4.5 h, and <3%–5% of stroke patients could be considered for this treatment. Even so, tPA only has a modest efficacy in improving functional outcome, and this drug is associated with a 10-fold risk of causing symptomatic intracranial hemorrhage.[1],[2],[3] Knowledge about the cause of stroke is limited, and the only established clinical management was to feed and care for the patient until the attack ran its course. The need for intervention is great because of the seriousness and prevalence of this disorder.

Ischemic Stroke

A stroke occurs when the blood supply to a part of the brain is suddenly interrupted. Brain cells die when they no longer receive oxygen and nutrients from the blood or there is sudden bleeding into or around the brain (NIH, NINDS). There are two forms of stroke: (1) ischemic blockage of a blood vessel supplying the brain and (2) hemorrhagic bleeding into or around the brain. Ischemic stroke is further divided into embolic stroke, thrombotic stroke, and stenosis according to the source of clots, while hemorrhagic stroke is subdivided into intracerebral hemorrhage and subarachnoid hemorrhage due to the location of aneurysm, vessel wall break, or arteriovenous malformation. In addition, transient ischemia attacks (TIAs or mini-stroke) start just like a stroke but then resolve leaving no noticeable symptom or deficits. Normally, the duration of TIA is within minutes, but symptom goes away within an hour – a prelude for stroke.

The incidence is ~85% for ischemic stroke and ~15% for hemorrhagic stroke in industrial countries. In this review, we focus on studies related to ischemic stroke (or cerebral ischemia), which triggers a complex series of biochemical and molecular reactions that impair the neurologic functions by cellular integrity breakdown, cell death, and infarction. The hypothetical cellular and molecular mechanisms underlying the ischemic stroke induced cell death including (1) energy failure (adenosine triphosphate [ATP] depletion), (2) lactic acidosis, (3) excitatory amino acid toxicity, (4) calcium overload, (5) oxidative stress, (6) inflammation, and (7) apoptosis. Noteworthy, it depends on the intensity and duration of ischemic insult; these events occur in an overlapping and sometimes interconvertible manner.[3],[4],[5]

An impressive amount of neuroprotective agents has been identified in experimental stroke. Despite major advances in the understanding of disease etiology, none of them proved efficient in clinical trials.[6],[7] There is a general consensus that an effective treatment requires the ability to interact with not one, but multiple pathophysiological cascades at different levels that induced by the insult – cocktail therapy.[8],[9] Luckily, recent progress in the field of epigenetics revealed that epigenetic modifications had influence on many known pathways involved in the complex course of ischemic disease develop. The fact that epigenetic molecules, by altering transcriptional regulation, may simultaneously act on different levels of ischemic brain injury makes them promising candidates for clinical use.[10],[11],[12],[13],[14],[15]

Epigenetic Regulations in Cerebral Ischemia

Epigenetic modifications

A chromosome is a deoxyribonucleic acid (DNA) molecule with part or all of the genetic material (genome) of an organism. Chromosomes in eukaryotes are composed of chromatin fiber. Nucleosomes are the basic unit of chromatin, which are composed of 146 base pairs (bp) stretch of genomic DNA wrapped around an octamer of core histone proteins that made up of two sets of H2A, H2B, H3, and H4. Between two nucleosome cores is a 38–53 bp linker DNA which is associated with H1 protein. During embryonic development, the term epigenesis refers to a set of mechanisms that resides above the level of genes, such as identical genes can be differentially expressed in different cell types and contexts to determine cell fate. However, epigenetics refers to a wide range of heritable changes in gene expression that occur in response to environmental influences and that do not result from alterations in the DNA sequence (Wikipedia). These alterations typically arise owing to DNA methylation, histone posttranslational modifications (PTMs), and changes in nucleosome positioning; these processes are collectively referred to by the broad term “chromatin remodeling.” Recent studies also identified histone variants, microRNAs (miRNAs), and long noncoding RNAs as additional epigenetic mechanisms.[11],[16],[17] DNA methylation and histone acetylation have been extensively studied than other mechanisms, which will be the focus in this review.

Deoxyribonucleic acid methylation

DNA methylation is essential for normal development and is associated with a number of physiological and pathological processes, including aging and carcinogenesis. DNA methylation was carried out by DNA methyltransferases (DNMTs), which transfer methyl groups from S-adenosyl-l-methionine to the 5-position carbon in cytosines within DNA to generate 5-methylcytosine. Three active DNMTs have been identified in mammals. They are DNMT1, DNMT3a, and DNMT3b. DNMT3a and DNMT3b are expressed mainly in the early embryo development to build up methylation pattern (de novo methyltransferases). DNMT1 is the most abundant DNMT in the mammalian cells which (1) maintain the methylation pattern established by DNMT3a and DNMT3b and (2) catalyze the transfer of methyl group to specific CpG structure in DNA. A fourth enzyme previously known as DNMT2 is a RNA, methyltransferase which has been changed to tRNA aspartic acid methyltransferase 1 to better reflect its biological function. In addition, DNMT3L interacts with DNMT3a/DNMT3b, is critical for DNA methylation, but has no methylation capability. Although this methyl group transfer was originally thought to be irreversible, evidence has revealed that DNA can rapidly undergo demethylation by a recent discovered family of 10–11 translocation methylcytosine dioxygenase proteins (TET1, TET2, and TET3), which catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine. The hydroxylmethyl moiety is labile and can rapidly regenerate unmethylated cytosines, which in turn activate genes. In general, DNA methylation occurs at CpG-rich promoter region which acts to suppress gene transcription via (1) physically hinder the binding of transcriptional proteins to the gene and/or (2) attracting the binding of methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases (HDACs) and other chromatin remodeling proteins that can modify histones, thereby forming compact, inactive chromatin, termed heterochromatin [Figure 1].[11],[18]

Ischemia leads to considerable alterations in gene expression; an overall increasing in the transcriptional repression is observed together with the corresponding increasing in the silencing epigenetic marks. The global amount of DNA methylation rises after an ischemic insult, and this increase correlates with augmented brain injury. Furthermore, analyzing the global methylation pattern of stroke patients with an epigenome-wide association study approach suggested that DNA methylation is associated with the risk of stroke, stroke recurrence, and functional outcome after stroke.[12],[15],[19],[20],[21] However, there were no global methylation differences in patients with different subtypes of ischemic stroke: large artery atherosclerosis, small artery disease, and cardio aortic embolism.[22] Furthermore, manipulation of transcription on the epigenetic level can yield a protective state. It has, for example, been demonstrated that pharmacological inhibition of DNA methylation ameliorates neurologic outcome in a rodent model of ischemia. Further, mice expressing reduced levels of DNMT1 in the postmitotic neurons are protected from ischemic brain injury.[15],[19],[21] However, no protective effect was observed in neuron-DNMT1 null mice.[23] The rationale under this discrepancy is not presently known. It is interested to note that during acute stroke, matrix metalloproteinases (MMPs) promote blood–brain barrier (BBB) leakage, edema, and hemorrhage; however, at delayed recovery phases, MMPs mediate beneficial plasticity and remodeling of the brain matrix.[24] In addition, inhibition of thrombospondin (TSP) is needed for postischemic angiogenesis; however, TSP is also required for the development of new synapses at recovery phase.[25],[26],[27] It is conceivable that individual gene may serve multiple roles at different phases/cells as stroke runs its course. In addition, besides DNA methylation in the promoter region, DNA methylation is also noted in the body of highly transcribed genes. For example, methylation at the CpG-rich introns 1 and 3 of interleukin 6 (IL-6) increased, not decreased, pro-inflammatory IL-6 generations.[28] Last but not the least, not all genes were hyper-methylated, DNA methylation in the NKCC1 promoter was decreased, and increased expression of NKCC1 was noted in the postischemic neurons, which is responsible for brain infarction.[29] The mechanisms and functions underlying DNA methylation are far more complicated than we thought, which deserve more attentions.

Histone acetylation

Histones

Histones are highly alkaline and conserved proteins found in the eukaryotic cell nuclei, which act as spools winding DNA to form nucleosomes. Changes in histone sequences mostly are lethal. Five major families of histones exist: H1, H2A, H2B, H3, and H4. Histones H2A, H2B, H3, and H4 are known as the core histones forming an octameric nucleosome core which was wrapped by ~146 bp DNA; while histone H1 is known as the linker histone, which binds the nucleosome at the entry and exit sites of the DNA, with approximately 38–53 bp DNA separating each pair of nucleosomes. All histones have a highly positively charged N-terminus with many lysine and arginine residues which facilitating DNA–histone interactions. Histones are subject to PTMs by enzymes primarily on their N-terminal tails. Each octameric nucleosome core has a central domain and several unstructured amino-terminal tails that contain more than 100 PTM sites for acetylation (Ac), methylation (Me), phosphorylation (P), ubiquitylation, sumoylation, deamination, O-GlcNAcylation, and poly (ADP) ribosylation. These marks are added by chromatin-remodeling enzymes known as “writers” and are removed by enzymes known as “erasers.” Distinct, site-specific PTMs of core histone proteins act sequentially or combinatorially to create a “histone code” that is recognized by proteins known as “readers.” Readers recognize epigenetic marks on core histones through specialized motifs and are thereby recruited to the promoters of target genes. Histone modifications act in diverse biological processes such as gene regulation, DNA repair, chromosome condensation (mitosis), and spermatogenesis (meiosis).[11],[30] Histone acetylation will be the main focus in this section, since it is the most well-studied PTM. In addition, genes encoding the above replication-dependent canonical histones are typically clustered along the chromosome, lack introns, and use a stem loop structure at the 3'end instead of a polyA tail, whereas genes encoding replication-independent histone variants are usually not clustered and have introns as well as their mRNAs are regulated with polyA tails and their function is beginning to be understood.

Acetylation and deacetylation

Acetylation refers to the process of introducing an acetyl group (CH3−C[=O]−) into a compound, namely the substitution of an acetyl group for an active hydrogen atom. Protein acetylation is an important modification in cell biology, which occurs cotranslational (before released from the ribosome) and PTM of proteins, indicating that acetylation has a considerable impact on gene expression and metabolism. N-terminal acetylation is one of the most common cotranslational covalent modifications of proteins in eukaryotes, which catalyzed by a set of enzyme complexes, the N-terminal acetyltransferases (NATs). NATs transfer an acetyl group from acetyl-coenzyme A (Ac-CoA) to the α-amino group of the first amino acid residue of the protein. Different NATs are responsible for the acetylation of nascent protein N-terminal, and the acetylation was found to be irreversible so far. To date, six different NATs have been found in humans – NatA to NatF. Each of these different enzyme complexes is specific for different amino acids or amino acid sequences.[31],[32]

Posttranslational acetylation of proteins is only observed on lysine reside, at its ε-amino group of side chain (R group), also using Ac-CoA as the acetyl group donor. Lysine acetylation and deacetylation are is catalyzed by enzymes with histone acetyl transferase (HAT) and the assistance of acetyl-CoA synthetase (ACS). Lysine acetylation also creates binding sites for bromodomain-containing proteins, such as HATs, methyltransferases, transcriptional coactivators, and ATP-dependent chromatin remodelers, within large protein complexes. These recruited proteins then act to alter chromatin structure and/or to promote transcription (histone code hypothesis).[33] In the field of epigenetics, histone acetylation-deacetylation has been shown to be important mechanisms in regulating gene transcription. However, nonhistone proteins, such as P53, tubulin, and NF-κB, are also regulated by HAT-mediated posttranslational acetylation, which play an important role in regulating enzyme activity and various signal transductions; thus, HAT is also named lysine acetyl transferases (KATs) nowadays to better reflect its biological function.[34],[35] Noteworthy, in a dynamic process, lysine and arginine residues can also be methylated by histone methyltransferases and demethylated by histone demethylases. The methylation marks can act as both, activating or repressive marks depending on the specific position of the residue. However, knowledge on histone methylation and its fundamental role in the transcriptional regulation is very primitive.[11]

Histone acetyl transferases/lysine acetyl transferases

HATs are enzymes that acetylate lysine amino acids on histone proteins by transferring an acetyl group from Ac-CoA to form ε-N-acetyl lysine [Figure 2]. In general, histone acetylation is linked to transcriptional activation. It was thought that acetylation of lysine neutralizes its positive charge, thus reducing affinity between histone and (negatively charged) DNA, which renders DNA more accessible to transcription factors (TFs). Nevertheless, recent studies revealed that lysine acetylation and other PTMs of histones generate binding sites for specific protein–protein interaction domains, such as the acetyl lysine-binding bromodomain, rather than through simply stabilizing or destabilizing the interaction between histone and DNA. There are two types of HATs which were divided according to their subcellular localization. Type A HATs are situated in the nucleus and type B HATs are situated in the cytoplasm. Type A HATs contain bromodomain, which recognizes and binds to lysine-acetylated histone, alone or as part of larger proteins complex, including other bromodomain-containing proteins, HDACs, MBD proteins, TFs, and co-activators. This binding is prerequisite for chromatin remodeling and gene expression. Based on the sequence and/or structure of the N-and C-terminal regions, type A HATs can be further categorized into five subfamilies: (1) general control nonderepressible-related acetyl transferases family including GCN5, PCAF, and ELP3; (2) p300/CREB-binding protein (CBP) family including p300 and CBP; (3) MOZ, YBF2/SAS3, SAS2, and TIP60 protein (MYST) family including MYST1, MYST2, MYST3, MYSY4, and tat-interacting protein 60 kDa (TIP60); (4) basal TF family including TFIIIC and TAF1; and (5) nuclear receptor cofactors family including SRC and ACTR/NCOA3. Type B HATs are different from type A HATs because type B HATs (1) do not have bromodomain; (2) recognize newly synthesized unacetylated histone to build up de novo acetylation pattern; and (3) play no role in chromatin remodeling and gene expression. In addition, the residues in the active site of type B are distinct from those of type A, suggesting different catalytic mechanism for acetylation. Knowledge on type B HATs is relatively less than type A. So far, only HAT1, HAT2, Rtt109, HatB3.1, and HAT4 have been reported.[33],[36]

It is known that ATP level was decreased in and around ischemic core, which led to subsequent decrease in levels of (1) acetyl-CoA, which is necessary for HATs to acetylate histone, and (2) phosphorylated HATs, which are required for optimum enzyme activity of HATs.[37] Thus, ischemia might inhibit HATs activity. Indeed, global transcriptional repression upon ischemia is accompanied by a decrease on histone H4/H3 acetylation. Consistently, HAT CBP is important in neuron survival following cerebral ischemia.[38] In contrast, genetic inhibition of HAT CBP/p300 yields neuroprotective effects after experimental global cerebral ischemia.[10],[15],[39] On the other hand, others also reported that HAT activity seems not significantly altered by cerebral ischemia.[40] The exact role of HATs in the pathogenesis of stroke deserves further investigation.

Histone deacetylases

HDACs are a class of enzymes, conserved in the evolution from yeast to plants and animals, which remove acetyl groups (O=C-CH3) from an ε-N-acetyl lysine amino acid on a histone, allowing the histones to wind the DNA more tightly [Figure 2]. Its action is opposite to that of HATs. HDACs are now also named lysine deacetylases to describe their function rather than their target, since they also remove acetyl groups from nonhistone proteins. These include a variety of different TFs, structural proteins, ion channels, receptors, and enzymes. Deacetylation of these proteins can have dramatic effects on their function, stability, subcellular localization, as well as their interactions with other proteins.[41],[42]

In humans, there are 18 HDAC subtypes that have been divided into four separate classes based on sequence, structural, and functional homology. Class I, II, and IV are considered “classical” HDACs whose activities are inhibited by trichostatin A (TSA) and have a zinc-dependent active site, whereas class III enzymes are a family of NAD+-dependent proteins known as sirtuins and are not affected by TSA. Within the class I HDACs, HDAC1, 2, and 3 are found primarily in the nucleus, whereas HDAC8 is found in both the nucleus and the cytoplasm and is also membrane associated. Class II HDACs can shuttle between the nucleus and cytoplasm and are further divided into two groups: Class IIa – consisting of HDAC4, 5, 7 and 9; and class IIb – consisting of HDAC6 and 10. HDAC11 is the sole member of class IV, while despite sharing similar characteristics with HDACs in classes I and II, HDAC 11 is thought to differ in its physiological properties. All of the classical HDACs are expressed in the brain, albeit at different levels depending on the region, which will be the focus of this review.[42],[43],[44] In addition, there are seven sirtuins (SIRT1-7) in class III HDACs, which divided into four subclasses by phylogenetic analysis: subclass 1 – SIRT1-3; subclass 2 – SIRT4; subclass 3 – SIRT5; and subclass 4 – SIRT6 and SIRT7. It has been shown that SIRT1, 2, 6, and 7 were expressed in the nucleus; SIRT1, 2, and 5 were identified in the cytoplasm; and SIRT3, 4, and 5 were found in the mitochondria. A remarkable feature of sirtuins is that they have two enzymatic activities: mono-ADP-ribosyltransferase and HDAC. The class III enzymes are structurally unrelated to classes I and II and considered a separate type of enzyme having different mechanism of action which will not be covered by this review (Wikipedia).[42],[43],[44]

Recent studies indicated robust protective roles of HDAC inhibitors against ischemic stroke, which will be addressed in detailed later.[10],[11],[13],[14],[15]

Acetyl-coenzyme A synthetase

Acetylation and deacetylation occur in a dynamic process in cells. While the acetylation of lysine residues is carried out by HATs using an acetyl group stemming from acetyl-CoA, the removal of acetyl group is performed by HDACs. Both HATs and HDACs lack intrinsic DNA-binding activity and are recruited to target genes via their association with transcriptional activators and repressors, as well as their incorporation into large multiprotein complexes,[44] including ACS. ACSs (also known as acetate-CoA ligase and acetyl-activating enzyme) are enzymes that combine acetate and CoA to form acetyl-CoA at the expense of ATP (ATP + acetate + CoA ≥ AMP + pyrophosphate + acetyl-CoA) [Figure 2]. Eukaryotes typically have two isoforms of acetyl-CoA synthase, a cytosolic form involved in biosynthetic processes and a mitochondrial form primarily involved in energy generation. Although activity of ACS is usually associated with metabolic pathways, this enzyme also participates in gene expression by replenishing acetyl-CoA to HATs for histone acetylation. It has been shown that attenuation of hippocampal ACS2 expression impairs long-term spatial memory, a cognitive process that relies on histone acetylation, by defecting upregulation of memory-related neuronal genes that are prebound by ACS2.[45],[46] Intriguingly, acetylation renders the ACSs inactive, while deacetylation restores full of these ACSs. The essential lysine residue in the active site plays an important role in the regulation of activity. The lysine molecule can be deacetylated by another class of enzyme called sirtuins. In mammals, the cytoplasmic-nuclear synthetase (ACS1) is activated by SIRT1 while the mitochondrial synthetase (ACS2) is activated by SIRT3. Both SIRT1 and SIRT3 confer protection against ischemia.[47] It would be interested to see how ACS response to low ATP environment. The role and expression pattern of ACS following ischemic stroke remain to be studied.

Histone Deacetylases in Stroke

Neuroprotective and neuroregenerative effects, ranging from attenuation of cell death/infarct to the stimulation of repair/regeneration, upon pharmacological inhibition of histone deacetylation have been reported in experimental and clinical stroke. HDAC inhibitors have been shown to provide robust protection against: (1) excitotoxicity: by restore glutamate receptor 2 (GluR2) and inhibit REST; (2) oxidative stress: by increasing HSP70 and HO-1 while decreasing Keap1; (3) ER stress: by preserving Ca++ homeostasis; (4) apoptosis: by increasing bcl-2 and bcl-xl while decreasing bax; (5) inflammation: by decreasing NF-ĸB, IL-1, IL-6, TNFα, iNOS and COX; and (6) BBB breakdown/edema: by decreasing MMP and preserving ZO-1. HDAC inhibitors have been shown as well as to promote (1) angiogenesis: by increasing HIF1α and VEGF; (2) neurogenesis: by increasing BDNF; and (3) stem cell migration: by increasing SDF1α to dramatically reduce infarct volume and improve functional recovery after experimental cerebral ischemia.[10],[11],[13],[14],[15] Interestingly, in spite of promising bench findings on the application of HDAC inhibitors in stroke, evidence for beneficial effects in human patients is lacking as no phase III clinical trials with HDAC inhibitors have been conducted in stroke as yet (sources: clinicaltrials.gov, http://www.ncbi.nlm.nih.gov/pubmed/). A major limitation is the lack of isotype-specific HDAC inhibitors that are permeable to the BBB and display reduced side effects. In general, HDAC inhibitors act through binding into the active site pocket and chelation of the catalytic zinc-ion located at its base. However, due to the highly conserved nature of the enzymatic pocket, the most HDAC inhibitors act rather unselective and inhibit either all or at least several members of the HDAC family simultaneously. Furthermore, these unspecific HDAC inhibitors were associated with numerous adverse effects such as bone marrow depression, diarrhea, weight loss, taste disturbances, electrolyte changes, disordered clotting, fatigue, and cardiac arrhythmias in stroke Phase I/II clinical studies. Nonetheless, analyzed data from three clinical cohorts revealed that HDAC inhibitors are associated with reduced stroke risk after previous ischemic stroke.[48] In addition, HDAC inhibitors have been linked to global DNA demethylation and cancer, as well as regulating miRNA pathways, in which mechanisms still remain unknown.[49],[50],[51] Intriguingly, both neuroprotective and neurotoxic effects of HDACs were noted in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.[44] Therefore, the roles of specific isoforms in the pathogenesis of stroke deserve further investigation. In this section, we review current knowledge on the genetics, expression, and function of the 11 classical HDAC family members in stroke. Noteworthy, the expression of all HDAC1-11 has been reported in murine and human brain (Allen Human Brain Atlas More Details – http://human.brain-map.org/).[52],[53] The gene structures of murine HDACs are depicted in [Figure 3]a and [Figure 3]b.

These HDACs are expressed ubiquitously, localized predominantly to the nucleus and display high enzymatic activity toward histone substrates. They possess relatively simple structures, consisting of the conserved deacetylase domain with short amino- and carboxy-terminal extensions. HDAC1 and HDAC2 are nearly identical and are generally found together in repressive complexes, such as the sin3, NuRD, CoREST, and PRC2 complexes. HDAC3 is found in distinct complexes such as the N-CoR–SMRT complex, whereas no complex has been described for HDAC8.[54],[55]

Histone deacetylase 1

Knockout (KO) of Hdac1 is embryonic lethal due to severe proliferation defects and retardation in development.[56] Genome-wide association study (GWAS) revealed that Hdac1 polymorphism is associated with asthma.[57] Transient middle cerebral artery (MCA) occlusion induced progressive decrease in cortical mRNA level of HDAC1[58] and HDAC1 gain of function provided potent protection against DNA damage in a forebrain ischemia model.[59] Nonetheless, no change in the HDAC1 level was observed after MCA occlusion.[60] In addition, in primary cortical neurons, oxygen-glucose deprivation (OGD) led to transient induction of HDAC1 mRNA and apoptosis; this cell death was counteracted by HDAC1 siRNA;[61] HDAC1 siRNA treatment also reduced infract volume in a transient MCA occlusion model by the same group (unpublished observations). Consistently, HDAC1 overexpression restored infarct volume by induced M1 microglial polarization in transient MCA occlusion model.[62] The reason for the above controversial observations is not presently known; nevertheless, it is well documented that HDAC1 conferred both neuroprotective and neurotoxic effects in neurodegenerative diseases.[44]

Histone deacetylase 2

Hdac2-null mice are viable until perinatal period with multiple cardiac defects.[63] GWAS revealed that Hdac2 polymorphism is associated with bipolar disorder.[64] Transient MCA occlusion led to progressive decrease in cortical mRNA level of HDAC2[58] or no change in cortical HDAC2 level.[60] While robust induction of HDAC2 mRNA was noted in primary cortical neurons following OGD insult.[61] Intriguingly, delayed induction of HDAC2 in the peri-infarct cortex was also been reported, and suppression of this HDAC2, by knockdown or KO of Hdac2, promoted recovery of motor function via enhancing cells survival and reducing neuro-inflammation, whereas overexpressing HDAC2 worsened stroke-induced functional impairment of both wild-type (WT) and Hdac2 conditional KO mice.[65],[66] Furthermore, smaller infarcts are also observed in HDAC2 KO mice after transient MCA occlusion by minimized loss of Endo-B1, and normalized expression of Mfn2.[67] Consistently, HDAC2 heterozygous mouse showed less retina ischemic injury than WT mouse.[68]

Histone deacetylase 3

Hdac3-deletion mice are embryonic lethal due to defect in cell cycle and apoptosis in mouse embryonic fibroblasts.[69] Hdac3 polymorphism contributes to an increased risk of schizophrenia and type 2 diabetes.[70],[71] Interestingly, mutation in both Hdac1 and Hdac3 increases tumor recurrence in hepatocellular carcinoma (HCC) patients.[72] Marked induction of HDAC3 was noted in ischemic cortex and in hypoxic primary cortical neurons, and specific knockdown HDAC3 by siRNA increased cortical neuronal survival.[58],[61] In addition, HDAC3 siRNA pretreatment mimics the beneficial effect of ischemic preconditioning, in which calpain-mediated C-terminus cleavage of HDAC3 is responsible for the beneficial effect of ischemic preconditioning in vitro and in vivo.[73] Nevertheless, no change in the cortical HDAC3 level after MCA occlusion was also reported.[60]

Histone deacetylase 8

Global deletion of Hdac8 in mice leads to perinatal lethality due to skull instability.[74] Mutation in Hdac8 associated with Cornelia de Lange syndrome increases neural stem cells death and abnormal facial morphogenesis.[75],[76],[77] Cortical HDAC8 mRNA level was either decreased[58] or no change[60] following transient MCA occlusion, whereas was increased after photothrombotic infarction.[65] Correspondingly, in primary cortical neurons, OGD led to transient induction of HDAC8 mRNA.[61] The role of HDAC8 in stroke is not presently clear, nonetheless, increasing cell death was noted in neurons derived from Hdac8 KO mice upon oxidative insults.[51]

Class IIa histone deacetylase (histone deacetylase 4, 5, 7 and 9)

These HDACs show relatively restricted expression patterns: HDAC5 and HDAC9 are highly enriched in muscles, heart, and brain. HDAC4 is highly expressed in the brain and growth plates of the skeleton, and HDAC7 is enriched in the endothelial cells and thymocytes. Class IIa HDACs have large N-terminal extensions with conserved binding sites for the TF myocyte enhancer factor 2 (MEF2) and the chaperone protein 14-3-3, which render HDACs signal responsive. Following phosphorylation by kinases, such as calcium/calmodulin-dependent protein kinase (CaMK) and protein kinase D, these HDACs bind 14-3-3 and shuttle from the nucleus to the cytoplasm. In contrast to other HDACs, class IIa HDACs possess only minimal catalytic activity due to a conversion of tyrosine to histidine in the catalytic domain. In addition, class IIa HDACs have been shown to recruit class I HDACs through their C-terminal HDAC domain, which probably accounts for a portion of their repressive activity.[51],[54],[55]

Histone deacetylase 4

Hdac4-null mice display premature ossification of developing bones due to ectopic and early-onset chondrocyte hypertrophy, and no Hdac4-null mice survived to weaning.[78] GWAS revealed that Hdac4 polymorphism is associated with schizophrenia and childhood obesity.[79],[80] Transient MCA occlusion led to a marked decreased expression of HDAC4 in the ischemic cortex, which is mediated by NADPH oxidase, and the subsequent activation of HMGB1 and neuronal death.[60],[81],[82] However, no change in the level of HDAC4 was observed in the ischemic cortex following transient MCA occlusion[58],[83],[84] and primary cortical neuronal upon OGD insult.[61] On the other hand, it has been shown that stroke induced nuclear shuttling of HDAC4 in neurons and nuclear-restricted HDAC4 expression aggravated neuronal death.[84] This stroke-induced HDAC4 nuclear translocation was blocked by CaMKIV and HDAC4 siRNA protects neurons from OGD insult.[84] In contrast, increased nuclear HDAC4 is strongly associated with neuronal remodeling but not with neuronal cell death, suggesting a role for nuclear HDAC4 in promoting neuronal recovery after ischemic injury.[85] In addition, ischemia increased phosphorylation of HDAC4, however, was noted in the endothelial cell, which promoting poststroke angiogenesis via HIF-VEGF signaling in vitro and in vivo.[83] Therefore, the exact role of HDAC4 in the pathogenesis of stroke deserved further investigation.

Histone deacetylase 5

Mice lacking Hdac5 were viable, were fertile, and had no abnormalities at early ages, while developed unusually enlarged hearts and abnormal cardiac stress response with age. Mice lacking both Hdac5 and Hdac9 survive until perinatal periods for lethal ventricular septal defects and thin-walled myocardium.[86] Mutation in Hdac5 associated with treatment efficacy or clinical improvement for major depression[87] or osteoporosis.[88] Transient MCA occlusion induced progressive decrease in the cortical mRNA level of HDAC5.[58],[60] It is suggested that HDAC5 protected cells from death through reducing HMGB1 expression and release and its expression was reduced by NADPH oxidase. Although HDAC5 is able to translocate between the cytoplasm and the nucleus, unlike HDAC4, no nuclear shuttling was observed following transient MCA occlusion.[85] Interestingly, HDACs 4 and 5 are expressed in dendrites of neurons in the normal and ischemic cortex, suggesting additional function for HDAC4/5 in dendritic outgrowth which promoting neuronal recovery after ischemic injury.[85] In contrast, expression and activity of HDAC5 were upregulated following transient MCA occlusion. HDAC5 via inhibiting myocardin-related TF-A (MRTF-A)-mediated anti-neuronal apoptosis fostered brain infarct.[89] The reason for the above controversial observations is not presently known.

Histone deacetylase 7

Hdac7-deletion mice are embryonic lethal due to a failure in endothelial cell–cell adhesion and consequent dilatation and rupture of blood vessels. HADC7 is specifically expressed in the vascular endothelium during early embryogenesis.[90] HDAC7 levels were not significantly altered following transient MCA occlusion[58],[60] and in OGD-treated primary cortical neuron.[61] Up-to-date, no report has discussed the effect of HDAC7 upon experimental stroke or hypoxic insult in vitro. Nonetheless, a HDAC7-derived 7-amino acid peptide (splicing form of HDAC7) could increase vascular progenitor cell migration and differentiation to enhance angiogenesis and ameliorate vascular injury in hind limb ischemic model.[91] On the other hand, it has been shown that only HDAC7 is marked increased in PLS-stimulated macrophage, and overexpression HDAC7 promotes TLR4-dependent pro-inflammatory gene expression via HIF-1α-mediated transactivation.[92] Nonetheless, the role of HDAC7 in stroke remains to be studied.

Histone deacetylase 9

Mice lacking Hdac9 had phenotypes very similar to Hdac5-deficient mice. Mice lacking both Hdac5 and Hdac9 survive until perinatal periods for lethal ventricular septal defects and thin-walled myocardium.[86],[93] GWAS identifies a variant in HDAC9, with increased activity, associated with large vessel ischemic stroke[94],[95] and schizophrenia.[96] Cortical HDAC9 level was either decreased[58] or increased[60] following transient MCA occlusion, while no significantly alteration was noted in OGD-treated primary cortical neurons.[61] It has been shown that HDAC9 expression was upregulated in human atherosclerotic plaques to activate macrophages in atherosclerosis development.[97]In vivo gene silencing of HDAC9 in the brain reduced cerebral injury in experimental stroke via suppressing endothelial autophagy.[98] In addition, Hdac9-KO mice resulted in a smaller infarct volume and an improved neurological function after transient MCA occlusion, by inhibiting inflammation responses, as compared with WT mice.[99] Noteworthy, there are 29 human HDAC9 protein isoforms, compared with six murine isoforms, listed in the NCBI data bank [Table 1]. Why human has so many HDAC9 isoforms and the exact role of HDAC9 in the pathogenesis of stroke deserve further investigation.

HDAC6 is highly enriched in the heart, liver, kidney, and placenta and HDAC10 is highly expressed in the liver, spleen, and kidney. HDAC6 is the main cytoplasmic deacetylase in mammalian cells, whereas little is known about the functions of HDAC10. Among the targets directly deacetylated by HDAC6 are cytoskeletal proteins such as α-tubulin and cortactin, transmembrane proteins such as the interferon α receptor, and chaperones. HDAC6 is distinct from all other HDACs as it harbors two deacetylase domains and a C-terminal zinc finger for ubiquitin binding. Although HDAC10 also contains two deacetylase domains, the second one has no deacetylase activity.[54],[55]

Histone deacetylase 6

Hdac6-deleted mice are viable, have no significant defects, show increase in global tubulin acetylation, and fail to form stress granules (SGs).[100],[101] Hdac6-deficient mice exhibit hyperactivity, less anxiety, and antidepressant-like behavior.[102] Dopaminergic abnormalities were also noted in Hdac6-deficient mice, indicating an association with psychiatric disorders such as schizophrenia.[103] GWAS revealed that HDAC6 polymorphism may associate with male fertility traits in cattle, not human.[104] HDAC6 is a novel critical component of SGs involved in the stress response; it coordinates the formation of SGs by mediating the motor protein-driven movement of individual SG components along microtubules. HDAC6 level was induced in ischemic cortex following MCA occlusion,[58],[105] and inactivation or wipeout HDAC6 reduced infarct volume and rescued neurons from apoptotic death.[58],[106],[107],[108] In addition, hypoxic treatment also increased HDAC6 level in primary cortical neurons and HDAC6 siRNA treatment rescued neurons from apoptotic death.[58],[61] Intriguingly, HDAC6 level also increased in endothelial cell after hypoxia, demonstrating that HDAC6 is necessary for angiogenesis.[109] In contrast, no change in HDAC6 level were also reported following MCA occlusion.[60] It would be interested to see the results of Hdac6-KO mice upon ischemic stroke.

Histone deacetylase 10

Hdac10 KO mice were healthy. GWAS revealed that HDAC10 polymorphism is associated with the development of HCC among chronic HBV patients[110] and plausible schizophrenia.[70] HDAC10 has a key role in Foxp3+ Treg cells and is an important new target for therapeutic intervention in transplant rejection and autoimmune diseases.[111],[112] No significant alteration in the HDAC10 level was reported following both in vivo MCA occlusion[58],[60] and in vitro OGD in primary cortical neurons.[61] Its role in stroke has not been reported. Nonetheless, it has been shown that HDAC10 overexpression in human umbilical vein endothelial cells (HUVECs) promoted tube formation, whereas depletion of HDAC10 from HUVECs inhibited tube formation in vitro and in vivo.[113]

Class IV histone deacetylase (histone deacetylase 11)

HDAC11 is the sole member in class IV, expression of HDAC11 is enriched in the brain, heart, muscle, kidney, and testis, but little is known about its function. It is composed of a deacetylase domain that shows homology to class I and II HDAC domains, with small N- and C-terminal extensions. HDAC11 is the only HDAC that does not form complex with other proteins for its activity.[54],[55] Hdac11 KO mice are viable and demonstrate more myeloid-derived suppressor cells (MDSCs) and enhanced tumor growth kinetics as compared to WT control. As of yet, no disease or developmental aberrations have been reported in these mice.[114] GWAS revealed that Hdac11 polymorphism is associated with age-related macular degeneration[115] and plausible schizophrenia.[70] HDAC11 appears to function as a negative regulator of MDSCs expansion/function in vivo. MDSCs are capable of suppressing anti-tumor T-cell function in the tumor microenvironment and represent an imposing obstacle in the development of cancer immunotherapeutics. No significant change in the HDAC11 level was reported following both in vivo MCA occlusion[60] and in vitro OGD in primary cortical neurons.[61] In contrast, HDAC11 mRNA was transiently increased while protein was decreased significantly after MCA occlusion.[58] Its role in experimental stroke has not been reported. Nonetheless, in kidney, ischemia/reperfusion downregulated the expression and binding of HDAC11 to PAI-1 promoter, which resulted in enhanced expression of PAI-1 and injury.[116] Highlights of this section are outlined in [Table 2].

Stroke is a devastating disease, yet no effective remedies are available to treat this disease. Luckily, recent progress in the research of pan-HDAC inhibitors opened a new avenue for treating stroke; however, lacking isotype specificity and adverse effects rendered their application in stroke patients. Currently, developing highly specific inhibitors is the mainstream research; however, it remains to be examined whether selective HDAC inhibitors will be equally effective as pan-HDAC inhibitors but with reduced side effects. It is worth noting that most pan-HDAC inhibitors are targeting both class I and II HDACs. An alternative approach is to regulate their gene activities since most of them were fluctuated upon hypoxic/ischemic insult. Unfortunately, mice mostly died if deficient in these evolutionary conserved enzymes which make research on their gene regulation rather difficult. The current review revealed that class I HDACs KO mice are lethal, either embryonic or perinatal; class IIa HDACs KO mice ranging from lethal to viable; class IIb HDACs KO mice are viable; while class IV HDAC (HDAC11) KO mice are health with no known developmental aberrations. Among them, both HDAC5 and HDAC9 KO mice are viable and developed cardiac defect with age. Excitingly, GWAS uncovered that a HDAC9 variant, the only HDAC, is associated with ischemic stroke. Consistently, HDAC9 KO mice showed less inflammation and reduced infarct upon ischemic stroke. Nevertheless, it remains to be studied the necessity for the presence of 29 human HDAC9 isoforms. In addition, epidemiological studies showed that HDAC5 was not a risk factor for stroke, although HDAC5 and HDAC9 shared redundant function. In addition, the role of the remaining HDACs in stroke was either not conclusive due to lethality or await to be validated in KO mice. Noteworthy, HDAC7 KO mice are embryonic lethal due to blood vessel rupture. It has been shown that HDAC7 is specifically expressed in endothelium. Intriguingly, the results of GWAS further showed that aberrant HDAC9 activity was mainly in the atherosclerotic plaques – immune cells. Along the line, HDAC6 and HDAC10 have been linked to function of Treg cell. This brings up an important issue that HDACs may serve different roles within various brain cell types and responses differently upon ischemic insult. For example, HDAC4 is harmful to neuron but promoting angiogenesis in the endothelial cells upon ischemic insult. This might in part, if not all, explain the inconsistent results obtained from different laboratories. Thus, cell-type-specific gene KO is a must to decipher its role in response to ischemic insult. In addition, many HDACs require multi-protein complexes or interaction with other HDACs for full enzymatic activity; multiple KO line probably better reflect the true intracellular situation. In view of the recent advances in CRISPR-CAS technology, it should not be a problem to generate conditional KO mice as needed. It is clear that the physiological functions of HDACs and their response to ischemic insult are far more complicated than we thoughts, which warranted further investigation. Advances in the physiological and pathological function of HDACs will not only enhance our knowledge on the underlying epigenetic mechanism in stroke but also greatly improve clinical therapeutic for patients.